类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

近年来,因病毒侵害人类每年都要蒙受巨大的经济损失和社会损失。犬肾细胞MDCK以其具有的培养容易、增殖快、流感病毒感染效率高等特点,成为适用于流感病毒疫苗生产的重要细胞系之一。以MDCK细胞为研究对象,在自制无血清培养基中成功实现了MDCK细胞从有血清培养到无血清培养的驯化;并通过单细胞悬浮培养驯化过程实现了MDCK细胞的无血清单细胞悬浮培养,获得了适于无血清单细胞悬浮生长的ssf-MDCK细胞株,无血清单细胞悬浮批培养最大活细胞密度可达3.81×106 cells/mL,最大比生长速率可达0.056 h?     

Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines

Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID₅₀) titers of the two cell lines reached 4.51×10⁶ cells/mL, 2.94Log₁₀(HAU/50 μL) and 8.49Log₁₀(virions/mL), and 5.97×10⁶ cells/mL, 3.88Log₁₀(HAU/50 μL), and 10.34Log₁₀(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.     

犬肾细胞MDCK无血清贴壁及单细胞悬浮培养

旨在挖掘用于鉴定金黄色葡萄球菌的高特异性靶点及其PCR检测引物。采用C++语言编程,以金黄色葡萄球菌Staphylococcus aureus MRSA 252基因组编码序列为对象,对2 656个可编码区进行分析,获得特异性靶点序列,并设计PCR扩增引物。对包括葡萄球菌属11个种及其他细菌属在内的共计137株细菌验证引物特异性,筛选获得9个DNA序列,并设计了4对引物。经验证2对引物的特异性较好,其中引物SA3的基因组DNA检测限为13.7 fg/μL,菌体检测限为9.25×102 CFU/mL。结果验证     

Serum suppresses the expression of hormonally induced functions in cultured granulosa cells

Growth and function of primary cultures of granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were compared in serum-containing and serum-free media. In serum-free medium (1:1 mixture of DMEM:F-12) supplemented with insulin, hydrocortisone, transferrin and fibronectin (4F medium), the cells remained healthy and steroidogenically responsive for at least 60 days in culture. The growth profile of the granulosa cells in 4F medium was similar to that obtained in serum-containing medium. In both media cell proliferation did not exceed more than one cell doubling. DMEM:F-12 alone did not support the cell viability. Upon FSH stimulation, the cells produced 25 fold more progestin and estrogen per cell in 4F medium than in medium supplemented with 5% serum. This effect was not directly related to serum proteins which mediate cell adhesion since cells cultured in dishes precoated with serum remained steroidogenically responsive to FSH. Cholera toxin and Bt₂-cAMP readily stimulated progestin production in the presence of serum. The inhibitory effect of serum was not reversed by adding the four factors to serum-containing medium. The factors were essential for the FSH-induced steroidogenesis in serum-free medium. After four days of incubation in 4F medium, the cells showed a transient loss of their ability to produce progestin in response to FSH. In both 4F medium as well as in serum-containing medium, the cells regained their hormonal responsiveness after 35 days in culture. Since the loss of hormonal responsiveness occurred at the same time as growth was initiated in the cultures, it is suggested that the FSH-induced steroidogenesis is negatively controlled by growth-related processes.     

Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.     

Serum-free media for cultures of primitive and mature hematopoietic cells

The in vitro culture of human hematopoietic cells has many research and therapeutic applications. Traditionally, human hematopoietic cultures have been conducted using serum-containing media. The disadvantages inherent in the use of serum could be eliminated by the use of serum-free media. In this review, we summarize and discuss the current status of serum-free media for both mature and immature human hematopoietic cells. The mature hematopoietic cells discussed are of lymphoid (e.g., lymphokine activated killer cells and tumor infiltrating lymphocytes) and myeloid origin (e.g., monocytes/macrophages). The cultures of immature hematopoietic cells discussed are clonogenic and long-term cultures. In addition, we briefly review the types of human hematopoietic cells, their clinical applications, and the basic strategies and components used to formulate serum-free media, Finally, we outline future requirements and directions in the development of serum-free media for primitive hematopoietic cells.     

Factors controlling embryonic heart cell proliferation in serum-free synthetic media

Summary Embryonic chick cardiac cell cultures, plated on collagen-coated dishes, containing serum-free synthetic media proliferate actively. The basic medium contained Ham's F12 nutrient mixture, fetuin, ascorbic acid, and bovine serum albumin. This medium was supplemented with various combinations of factors; endothelial cell growth supplement (ECGS), epidermal growth factor (EGF), insulin (I), transferrin (T), selenium (S), hydrocortisone, and thyroxine or supplemented alone. Basic medium supplemented with ECGS alone contributes to the highest final cell density among all other factors used in various combinations or alone. The final cell density of the control culture with 2% fetal bovine serum was higher than those of all experimental cultures and an additional control culture grown in the basic medium. Combinations of factors without ECGS do not promote significant cell proliferation. Thyroxine is required to induce optimal differentiation and contractility of cardiac myocytes in vitro. Fibronectin and laminin did not show any more influence than collagen did on the growth and maintenance of cardiac myocytes in serum-free media. The proportion of cardiac muscle cells in ECGS-containing media was higher than those in other experimental media and control media with the exception of ECGS and ITS-containing medium that showed lower proportion of cardiac myocytes than that of serum-containing medium on Days 3 and 5. The profiles of incorporation of [³H]thymidine into DNA of heart cells in experimental and control cultures showed a peak in incorporation values within the first week of culture and subsequently declined. Autoradiography studies revealed that cardiac myocytes in culture supplemented with ECGS alone attained a peak in labeling index on Day 1 with approximately 62% labeled cells. Subsequently, the labeling indices declined. Cardiac myocytes grown in media without ECGS showed significantly lower labeling indices than those in ECGS-containing media. This study has demonstrated the influence of ECGS, EGF and ITS in promoting the growth of cardiac myocytes and also in contributing to the maintenance of contractile cardiac myocytes in serum-free, long-term culture. The influence of ECGS on heart cell proliferation is considered to be superior to that of EGF and ITS. This study was supported in part by a grant HL-25482 from the National Heart Lung and Blood Institute and a grant from the American Heart Association of Michigan.     

钙离子对293细胞结团和生长的影响

分别在有血清和无血清条件下、方瓶和转瓶中考察了Ca²⁺ 对2 93细胞结团和生长的影响。通过实验发现,Ca²⁺ 浓度在0 1～1 0mmol L范围内对2 93细胞的贴壁和结团性质有显著影响,而对生长影响不大。结果表明:有血清贴壁培养时,较高的Ca²⁺ 浓度有利于细胞贴壁;无血清悬浮培养中,Ca²⁺ 浓度越高,细胞结团越严重,细胞结团达到平衡后的平均粒径(D ,μm)与Ca²⁺ 浓度(c,mmol L)在0.1～0.5mmol L范围内可用一次函数D =58.65c +16.96描述,细胞结团尺寸是可调控的;而细胞在不同的Ca²⁺ 浓度下有相似的生长规律。     

Delayed dedifferentiation and retention of properties in dissociated adult skeletal muscle fibers in vitro

Adult skeletal muscle fibers can be isolated and cultured but tend to dedifferentiate and sprout with time in culture. We examined isolated adult mouse flexor digitorum brevis muscle fibers under various culture conditions by monitoring maintenance of the same fibers at 2-d intervals using survival analysis. Fibers plated on laminin and cultured in serum-free media did not show sprouting and exhibited significantly (P < 0.0001) longer survival (median survival time, T(50) = 10.2 d) than fibers in serum-containing media (T(50) = 3.3 d). Cell proliferation was markedly suppressed in serum-free cultures. Multiple or delayed Ca(2+) transients in response to brief field stimulation were often observed in dedifferentiated fibers after several d in serum-containing media but were not observed in fibers in serum-free media. The addition of cytosine arabinoside to serum-containing cultures did not prolong fiber survival (P = 0.39) and did not eliminate sprouting but did greatly suppress proliferation of nonmuscle cells. Fibers cultured in agarose gel with serum exhibited small, bud-like extensions but no sprouts and did not survive as long (T(50) = 6.2 d) as fibers plated on laminin and cultured in serum-free media (T(50) = 10.2 d) did. These results demonstrate that both morphological and physiological properties of fibers become modified in serum-containing media but can be retained by culturing without serum.     

Human glioma cell culture: two FCS-free media could be recommended for clinical use in immunotherapy

Immunotherapy, particularly active vaccination, may be developed as an effective and safe treatment modality for malignant gliomas, which continue to have a poor prognosis, despite advances in surgical techniques and adjuvant chemotherapy and radiotherapy. Since no glioma-specific tumor-associated antigens (TAAs) have been discovered, autologous tumor cells or well-established glioma cell lines could be used in future vaccination protocols to induce antitumour immunity against unknown TAAs. One obstacle for clinical use of these tumour cell vaccines is related to foetal calf serum (FCS). Efforts are currently being directed toward developing FCS-free media and serum-free alternatives to culture these cell vaccines. In this study, a medium containing human serum and one serum-free medium (UltraCulture), supplemented or not with epidermal growth factor, were tested on morphology, survival, DNA content and TAA expression of human glioma cell lines and glioma biopsy primary cultures. Their effects were compared on FCS-containing medium. Results show that, whatever the medium used, no significant variations in morphology and survival were observed. Furthermore, human serum-containing medium or UltraCulture preserved at early passage cultures the cell population of interest present in the biopsies before culture. In addition, the expression profile of eight TAAs was similar between these media. These data indicate that human serum-containing medium and UltraCulture serum-free medium could be promising candidates to produce tumour-cell vaccines.     

Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media

The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.     

黄牛表皮细胞分离与体外培养最适条件的探索

为了建立黄牛表皮细胞分离与体外培养的最适条件,比较了组织块法与单细胞悬液法、不同蛋白酶(胰蛋白酶和分离酶)的消化以及有无血清培养基对细胞生长的影响,以克隆形成率来检测细胞生长和存活情况。结果证明：采用分离酶分离表皮细胞进行无血清培养是黄牛表皮细胞体外培养的最适条件。     

Rabbit chondrocytes maintained in serum-free medium : I. Synthesis and secretion of hydrodynamically-small proteoglycans

The biosynthesis of sulfated proteoglycan in vitro by rabbit articular chondrocytes in first passage monolayer culture maintained in fetal bovine serum (FBS) or in serum-free conditions was compared. Neosynthesized proteoglycan in the culture medium in the most dense fraction of an associative CsCl density gradient (fraction dAl) declined with increasing time under serum-free conditions, but not when cells were maintained in the presence of serum. After one day, the major peak of incorporated 35SO4 in medium fraction dAl eluted as a retarded peak (Kav 0.28) on Sepharose CL-2B, whether cells were maintained under serum-free or serum-containing conditions. The hydrodynamic size of proteoglycan monomer fraction dAlDl obtained after one day of exposure to serum-free culture media was smaller than dAlDl from serum-containing cultures. The hydrodynamic size of dAlDl obtained from serum-free culture media became even progressively smaller after 2 and 3 days' exposure to these conditions. Hydrodynamically small sulfated proteoglycans were identified in the cell-associated dAlDl fraction as early as one day after switching chondrocytes from serum-containing to serum-free medium. Culture medium fraction dAlDl from serum-free culture medium aggregated poorly when incubated with human hyaluronic acid (HA) in the presence of bovine link protein or when dialysed against bovine nasal cartilage proteoglycan aggregate. Proteoglycan monomer from serum-containing medium reaggregated more efficiently under both conditions. No change in the size of glycosaminoglycan chains was seen in the smaller proteoglycan subpopulations, nor was there any indication of marked changes in the glycosaminoglycan types.     

A serum substitute for fed-batch culturing of hybridoma cells

We developed a substitute for serum to produce fed-batch cultures of hybridoma cells in serum-free medium and confirmed that the cells could be successfully cultivated this way. Our substitute consisted of 12 components. The specific production rates of lactate and ammonia, which are harmful byproducts from the cells, were significantly reduced compared with a conventional serum-containing batch culture. This reduction led to a higher cell concentration and a longer production lifetime. As a result, the final concentration of monoclonal antibody was 400 mg/L, or five times greater than that in the conventional serum-containing batch culture. The developed substitute is expected to enable fed-batch cultivation in a serum-free condition.     

Evaluation of the new serum-free medium (MDSS2) for the production of different biologicals: Use of various cell lines

The presence of serum in cell culture raises safety problems for the production of biologicals, thus a new serum-free medium (MDSS2) was developed. The evaluation of this medium for the growth of different cell lines (BHK-21 C13, BSR and Vero) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static (as monolayer) as well as in agitated systems (in suspension in spinner and perfusion reactors). BHK-21 and BSR cells grew as aggregate cultures and could proliferate in both static and agitated culture systems. Vero cells stayed attached to a substrate and proliferated equally in static and in agitated microcarrier-culture systems. The cell densities obtained with BHK-21 cells depended only on the culture system used. They ranged from 2–3×10⁶ to 6–12×10⁶ cells per ml for static batch and perfusion reactor cultures respectively. The cell concentration was 3 to 6 times higher than in classical cultures performed in serum-containing medium. The cell densities obtained with Vero cells were indistinguishable from those obtained in serum-containing medium, whatever the cell culture system used. These cell lines have been used for the production of rabies virus. With respect to BHK-21 and BSR, similar production rates of rabies glycoprotein have been found as in the standard roller bottle process. The production of rabies virus and of viral glycoprotein by Vero cells cultivated in serum-free medium was augmented 1.5-fold and 2.5-fold, respectively, when compared to serum-containing medium.A recombinant BHK-21 cell line, producing human IL-2, can also proliferate in MDSS2, after addition of insulin. The specific IL-2 production rate was augmented 3–4 fold in comparison to serum-containing medium.For the cells tested, the MDSS2 serum-free medium is a good growth and production medium. Its use for cultivating other cell lines and/or for the production of other biologicals is discussed.     

Migration inhibition of an epithelial cell line by s-Con A and the effect on its invasiveness

In an attempt to delineate the role of tumor-cell motility in the process of invasion, we compared the migration of NBT II in a two-dimensional migration assay with its migration in a three-dimensional invasion assay. Both systems were maintained with and without succinylated concanavalin A (s-Con A) dissolved in the culture medium. This lectin has a reversible inhibitory effect on the migration of cells in vitro. The migration of NBT-II aggregates, seeded in flasks containing 200 micrograms/ml s-Con A or without s-Con A, was studied by time-lapse photomicrography. In the presence of s-Con A, migration was immediately stopped. When the treated medium was replaced by culture medium to which 10 mM alpha-methyl-mannose was added, the inhibition of migration was abolished. The invasive capacity of NBT II in the presence or absence of s-Con A was studied by confronting precultured fragments of 9- to 11-day-old embryonic chick heart (0.4 mm in diameter) with NBT-II aggregates (0.2 mm) made in the presence or absence of s-Con A. Light microscopy showed no difference in the extent of invasion. To demonstrate the presence of s-Con A in the invading tumor cells, immunoperoxidase staining for Con A was done. The treated cultures stained positively while the controls were negative. The data presented here question the correlation between tumor-cell motility in two-dimensional system and the invasive behavior of these cells in three dimensions, and implies that the ability or inability of cells to migrate on plastic does not necessarily reflect their invasiveness in vitro.     

Formulation and production of a blood-free and chemically defined virus production media for VERO cells

Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a slower doubling time in both two-dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.     

Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells

The necessity to perform serum-free cultures to produce recombinant glycoproteins generally requires an adaptation procedure of the cell line to new environmental conditions, which may therefore induce quantitative and qualitative effects on the product, particularly on its glycosylation. In previous studies, desialylation of EPO produced by CHO cells was shown to be dependent on the presence of serum in the medium. In this paper, to discriminate between the effects of the adaptation procedure to serum-free medium and the effects of the absence of serum on EPO production and glycosylation, adapted and non-adapted CHO cells were grown in serum-free and serum-containing media. The main kinetics of CHO cells were determined over batch processes as well as the glycosylation patterns of produced EPO by HPCE-LIF. A reversible decrease in EPO production was observed when cells were adapted to SFX-CHO^TM medium, as the same cells partially recovered their production capacity when cultivated in serum-containing medium or in the enriched SFM^TM serum-free medium. More interestingly, EPO desialylation that was not observed in both serum-free media was restored if the serum-independent cells were recultured in presence of serum. In the same way, while the serum-independent cells did not release a sialidase activity in both serum-free media, a significant activity was recovered when serum was added. In fact, the cell adaptation process to serum-free conditions did not specifically affect the sialidase release and the cellular mechanism of protein desialylation, which appeared to be mainly related to the presence of serum for both adapted and non-adapted cells.     

Culture of endocrine pancreatic cells in protein-free,chemically defined media

Summary Cell suspensions prepared by collagenase digestion of pancreata obtained from 21.5-d-old rat fetuses were preincubated in RPMI medium containing 10% fetal bovine serum (FBS), to ensure cell adhesion. Twenty hours later, this medium was replaced by a chemically defined medium. Dulbecco's modified Eagle's (DME)-F12 was used alone or supplemented with various combinations of transferrin, sodium selenite, or Ultroser G. The evolution of the culture and the islet ultrastructure were similar in defined and serum-containing media. However, in the defined medium, the neoformed islets seemed less numerous, and the fibroblast layer less dense, when compared to the RPMI+10% FBS control medium. At Day 7, in defined media, the total insulin content per dish was half that of control cultures. None of the tested additives improved the yield of the cultures. The fractional insulin release per day was elevated in defined media. In subsequent incubations, glucose and leucine stimulated insulin release in a way characteristic of these cells of fetal origin. The labeling index of islet cells cultured in DME-F12 reached 10.7%, which is not far from that observed in RPMI+10% FBS. Such a defined medium is useful to study B cell physiology, avoiding the possible interaction of serum components with substances to be tested. The support of the Fonds National de la Recherche Scientifique and the ‘Loterie Nationale’ of Belgium is also acknowledged.     

Stirred culture of peripheral and cord blood hematopoietic cells offers advantages over traditional static systems for clinically relevant applications

The ability to culture hematopoietic cells in readily characterizable and scalable stirred systems, combined with the capability to utilize serum-free medium, will aid the development of clinically attractive bioreactor systems for transplantation therapies. We thus examined the proliferation and differentiation characteristics of peripheral blood (PB) mononuclear cells (MNC), cord blood (CB) MNC, and PB CD34(+) cells in spinner flasks and (control) T-flask cultures in both serum-containing and serum-free media. Hematopoietic cultures initiated from all sources examined (PB MNC, CB MNC, and PB CD34(+) cells) grew well in spinner vessels with either serum-containing or serum-free medium. Culture proliferation in spinner flasks was dependent on both agitator design and RPM as well as on the establishment of critical inoculum densities (ID) in both serum-containing (2 x 10(5) MNC/mL) and serum-free (3 x 10(5) MNC/mL) media. Spinner flask culture of PB MNC in serum-containing medium provided superior expansion of total cells and colony-forming cells (CFC) at high ID (1.2 x 10(6) cells/mL) as compared to T-flask controls. Serum-free spinner culture was comparable, if not superior, to that observed in serum-containing medium. This is the first report of stirred culture of PB or CB MNC, as well as the first report of stirred CD34(+) cell culture. Additionally, this is the first account of serum-free stirred culture of hematopoietic cells from any source.